Spinal cord Injury is a problem affecting millions worldwide. A dominant hypothesis in regenerative neuroscience is that patients with spinal cord injury suffer from permanent functional deficits and paralysis because of neural damage, but also because of a limited capacity of CNS axons to regenerate and restore lost neuronal connectivity. According to this scheme, a primary cause of failed regeneration is a limited intrinsic ability of adult neurons to regrow injured axons and the growth-hostile environment of the damaged cord. Several critical molecules that impede axon regeneration in the injury territory have been identified and include myelin-derived growth-inhibitory proteins such as myelin associated-glycoprotein (MAG) and reactive astrocyte-produced chondroitin sulfate proteoglycans (CSPGs). Myelin and astrocyte-derived inhibitory signals ultimately converge on the actin and microtubule cytoskeleton, affecting their stability, dynamics, and ability to direct axonal growth. We have found that specifically targeting histone deacetylase 6 (HDAC6) in neurons, using both pharmacological inhibitors and knockdown methods, can overcome the inhibitory effects of MAG or CSPGs to axon growth, in vitro. Using microfluidic chambers that isolate axons from the neuronal cell bodies we have determined that local processes in the axon mediate this effect. Consistent with this, we have found that HDAC6 inhibition or knockdown does not increase histone acetylation (a canonical function of pan-HDAC inhibitors that target multiple HDAC isozymes) and that recovery of growth can occur in the presence of a transcriptional inhibitor. A primary, and non-nuclear, function of HDAC6 is the deacetylation of 1-tubulin lysine 40 and, in turn, the modulation of microtubule dynamics. Given the role of the microtubule in axon growth, we hypothesize that HDAC6 plays a role in mediating a cell's response to myelin and astrocyte derived inhibitory signals via 1- tubulin deacetylation and microtubule destabilization. In the Specific Aims of this Application, we will examine the function of HDAC6, as well as the 1-tubulin acetylating enzyme, Elp3, in growth-inhibited axons. We will examine the extent to which their activities modulate the acetylation level of 1-tubulin and the role of 1-tubulin deacetylation in microtubule destabilization and axonal regeneration failure. We also will test whether HDAC6 plays a role in axon regeneration failure in vivo and whether increasing 1-tubulin acetylation by HDAC inhibition enhances axonal regeneration after spinal cord injury. PUBLIC HEALTH RELEVANCE: Disability following spinal cord injury is attributed, in part, to a limited ability of central nervous system axons to regenerate. Contributing to this inability is the injury environment, where a number of growth-inhibitory proteins are known to be present (e.g., myelin associated-glycoprotein and chondroitin sulfate proteoglycans). Strategies that promote axons to re-grow in their presence are needed. Signals from these growth-inhibitory proteins ultimately converge on the actin and microtubule cytoskeleton, affecting their stability, dynamics, and ability to direct axonal growth. We have found that inhibiting histone deacetylase-6 (HDAC6), an enzyme responsible for the modulation of microtubules, appears to be an excellent, non-toxic therapeutic strategy for promoting regeneration in cell culture models. The proposed studies in this application will further define the mechanisms of HDAC6 inhibition-mediated axonal regeneration, as well as test its therapeutic efficacy in pre- clinical rodent models of spinal cord injury. Our findings in this project could lead to new treatments for spinal cord injury and other central nervous system injuries and disease.